EP2072617A1 - Method for producing dendritic cells - Google Patents

Method for producing dendritic cells Download PDF

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EP2072617A1
EP2072617A1 EP07450233A EP07450233A EP2072617A1 EP 2072617 A1 EP2072617 A1 EP 2072617A1 EP 07450233 A EP07450233 A EP 07450233A EP 07450233 A EP07450233 A EP 07450233A EP 2072617 A1 EP2072617 A1 EP 2072617A1
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Prior art keywords
immune
dcs
hours
dendritic cells
cd40l
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EP07450233A
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German (de)
English (en)
French (fr)
Inventor
Thomas Felzmann
Alexander Michael Dohnal.
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TRIMED BIOTECH GmbH
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TRIMED BIOTECH GmbH
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Priority to EP07450233A priority Critical patent/EP2072617A1/en
Priority to MX2010006434A priority patent/MX2010006434A/es
Priority to US12/747,759 priority patent/US9303246B2/en
Priority to CN200880120431.4A priority patent/CN101896601B/zh
Priority to EP08859543.4A priority patent/EP2222836B1/en
Priority to CA2709209A priority patent/CA2709209C/en
Priority to KR1020107011455A priority patent/KR101590199B1/ko
Priority to AU2008334914A priority patent/AU2008334914B9/en
Priority to JP2010537327A priority patent/JP5730577B2/ja
Priority to NZ585898A priority patent/NZ585898A/en
Priority to BRPI0819936-1A priority patent/BRPI0819936A2/pt
Priority to PCT/EP2008/010606 priority patent/WO2009074341A1/en
Priority to EA201000972A priority patent/EA023106B1/ru
Publication of EP2072617A1 publication Critical patent/EP2072617A1/en
Priority to ZA2010/03193A priority patent/ZA201003193B/en
Withdrawn legal-status Critical Current

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Definitions

  • the present invention relates to a method for producing and genetically engineering dendritic cells (DC).
  • DC dendritic cells
  • DC dendritic cell
  • IL interleukin
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • iDC immature DCs
  • iDCs are sentinels that sample their environment by taking up material from the extra cellular fluid as well as apoptotic bodies from physiologically dying cells, process, and present this material without co-stimulation in a tolerance-inducing form to T-lymphocytes.
  • the tolerance-inducing iDC phenotype may be considered the default status of DCs.
  • a danger signal that may be a pathogen associated molecular pattern (PAMP) transmitted by toll-like receptors (TLR), inflammatory cytokines, or T-lymphocyte derived signalling, most prominently mediated by CD40/CD40L interaction.
  • PAMP pathogen associated molecular pattern
  • TLR toll-like receptors
  • inflammatory cytokines cytokines
  • T-lymphocyte derived signalling T-lymphocyte derived signalling
  • IL-12 release ceases after about 24 hours indicating that the encounter between DCs and T-lymphocytes needs to take place within that time window to allow efficient type 1 polarisation and CTL activation.
  • the expression of co-stimulatory molecules reaches its maximum after 2 days. Since per definition a mature DC is characterised only phenotypically but not functionally by maximum expression of co-stimulatory molecules, the IL-12 releasing type 1 polarising DC is referred to as semi-mature (sm) DC.
  • the present invention relates to a method for producing dendritic cells comprising the steps of:
  • Genetically engineered IL-12 releasing DCs of step b) over-express at least one molecule capable of extending the T-lymphocyte stimulatory time window and/or entirely preventing its closing after 24 hours characterised by maintained secretion of longer than 24 hours by introducing nucleic acid molecules encoding for said at least one immune stimulatory molecule; or show an inhibited or down-regulated expression of at least one molecule that is involved in the normal developmental progress of a DC after exposure to any effective maturation stimulus from the T-lymphocyte stimulatory into a T-lymphocyte suppressive time window that starts opening 24 hours after maturation; and/or inhibit or prevent the expression of at least one molecule that fulfils a function in mediating T-lymphocyte suppression by DCs that have developed to assume an immune suppressive phenotype.
  • the dendritic cells of the present invention maximise T-lymphocyte stimulation, particularly CTL activation, by using genetic engineering to broaden the stimulatory time window of approximately 24 hours or entirely prevent closing of this stimulatory time window.
  • IL-10 or IDO other molecules that are involved in the immune suppressive function of the DCs that starts approximately 24 hours after exposure to any maturation stimulus (table 3 and 4) may be used. It will be these molecules that are preferably targeted in the manufacturing of genetically engineered immune stimulatory DCs. It is particularly preferred to use molecules that show a two-fold over expression in the presented DNA micro array data (table 3 and 4), more preferable an over-expression of at least six fold. The numbers given in tables 3 & 4 show the fold over-expression as indicated in the heading of the respective column.
  • T-lymphocyte suppressive DC immune medicine for the treatment of pathological over-activity of the immune system, e.g. in allergies or autoimmune diseases, as well as in stem cell and organ transplantation.
  • Immune suppression is physiologically mediated by a DC that has differentiated for more than 24 hours after exposure to any maturation stimulus.
  • the immune suppressive capacity of such a DC is enhanced by interfering with the expression of T-lymphocyte stimulatory molecules during the first 24 hours of DC differentiation; and/or by over-expressing molecules that confer T-lymphocyte suppression by genetically engineering the DC according to the strategies outlined above.
  • dendritic cells obtained with the method according to the present invention exhibit a broader stimulatory window (i.e. increased and prolonged expression of IL-12).
  • genetic engineering of a semi-mature (sm) DC - a DC in which the physiologic differentiation process is initiated by exposure to any maturation stimulus capable of triggering IL-12 secretion from DCs, but which, however, is removed preferably after two to twelve hours, more preferably after six hours - to over-express the CD40L molecule has the capacity to maintain its T-lymphocyte stimulatory capacity for at least 24 hours, preferably 48 hours, and even up to five or even ten days. It is furthermore preferred to culture such genetically engineered DCs in medium containing IFN- ⁇ .
  • Such DCs render smDCs by a typically six hour-exposure to a Toll-like receptor (TLR) ligand, preferably but not exclusively lipopolysaccharide (LPS), again preferably in the presence of IFN- ⁇ , - see Table 1 - and genetically engineered to over-express CD40L, assume a phenotype that is characterised by continued secretion of IL-12 for at least one, preferably three, and even up to five days and the maintenance of the immune stimulatory capacity in an allogeneic mixed leukocyte reaction (alloMLR) for at least 24 hours, preferably 48 hours, but up to five days.
  • TLR Toll-like receptor
  • LPS lipopolysaccharide
  • an initial maturation stimulus such as LPS/IFN- ⁇ needs to be applied to the DC in order to initiate the physiologic differentiation from iDCs into smDCs.
  • Other TLR ligands (table 1) may serve the same purpose as LPS; combinations of TLR ligands may give a stronger but not a qualitatively different signal. If the stimulatory potential of a T-lymphocyte stimulatory DC immune medicine is based only on the artificial manipulation of the gene transfer without the initial exposure to a TLR ligand mediated maturation stimulus (e.g.
  • a critical difference of the genetically engineered DC immune medicine according to the present invention to a DC immune medicine that is manufactured by only an exposure to LPS/IFN- ⁇ (smDC) is that for the latter it is critical that the smDC immune medicine is applied during the corresponding brief window of DC differentiation.
  • smDC LPS/IFN- ⁇
  • Such a stimulatory DC immune medicine has therefore to be applied early after exposure to the maturation stimulus, whereas the genetic engineering e.g. by over-expression of CD40L by broadening the immune stimulatory time window of DC differentiation, allows for a less time critical application.
  • the immune stimulatory time window of the older smDC immune medicine closes after 24 hours, whereas the novel genetically engineered DC immune medicine will maintain its T-lymphocyte stimulatory potential for at least one, preferably three, but up to five days longer.
  • a comparable concept holds true for a T-lymphocyte suppressive DC immune medicine.
  • the immature DCs first need to be exposed to a conventional maturation stimulus, such as LPS/IFN- ⁇ in order to initiate differentiation towards an mDC phenotype corresponding to the T-lymphocyte suppressive window of DC differentiation.
  • the genetic engineering to over-express T-lymphocyte suppressive molecules from the DC immune medicine may be done six hours and up to 48 hours after initiation of maturation by e.g. LPS/IFN- ⁇ .
  • TLR ligands Receptor Naturally occurring Synthetic analogues Fully synthetic small molecules Exogenous ligands TLR1 Not determined Triacyl lipopeptides - TLR2 Lipoproteins/lipopeptides Peptidoglycan Lipoteichoic acid Lipoarrabinomannan Atypical lipopolysaccharide Di- and triacyl-lipopeptides - TLR3 Double-stranded RNA PolyI:C - TLR4 Lipopolysaccharide HSP60 (Chlamydia pneumonie) LPS/lipid A mimetics, such as MLP Synthetic lipid A, E5564 TLR5 Bacterial flaggelin Discontinuous-13-aminoacid peptide - TLR6 Not determined Diacyl lipopeptides - TLR7 (G+U) rich single-stranded RNA (mouse only) Oligonucleotides Imidazole quinolines (imiquimod, resiquimod),
  • a T-lymphocyte suppressive DC immune medicine may be designed by knocking out the expression of molecules, such as CD40 or IL-12 or similar molecules, or by over-expressing molecules that confer T-lymphocyte suppression from the DC immune medicine.
  • the immune suppressive window of DC differentiation may be closed or made narrower or moved to a later time point.
  • the feasibility of this approach is demonstrated by knocking down the expression of molecules that interfere with T-lymphocyte activation by DCs.
  • IL-10 As a second example the expression of IL-10 was targeted, which is considered the prototypic immune suppressive molecule and which is expressed by DCs during the immune suppressive differentiation time window.
  • RNA interference is preferably used, but other technologies, such as the intracellular expression of single chain monoclonal antibodies or anti-sense RNA, may serve the same purpose.
  • over-expression of said molecules e.g. IDO or IL-10) or similar molecules may serve to design a T-lymphocyte suppressive DC immune medicine on the basis of pre-matured smDCs.
  • the structure and properties of a DC need to be described in a dynamic fashion that takes into consideration the developmental stages of a DC. Each of these stages may be characterised by the absence or presence of certain marker molecules. This also indicates that the molecular features of a DC depend on the specific stage of differentiation of this DC and the conditions that caused a DC to assume a certain differentiation pathway.
  • the developmental plasticity of a DC also explains why it is advantageous to use what is called a semi-mature type 1 DC (smDC1) ("T cell activating dendritic cells characterised by the release of interleukin 12").
  • smDC1 semi-mature type 1 DC
  • IL-12 acts via a specific receptor on helper T-lymphocytes and causes them to assume a Th1 phenotype, resulting in the support of cytolytic immunity.
  • the DCs are preferably inoculated into the organism (e.g. human) at an early time point during the immune stimulatory window. It is particularly preferred that said DCs are injected 6 hours after the maturation stimulus.
  • inoculation is associated with removal of the DC culture medium that contains the dendritic cell maturation agent (e.g. stimulatory molecules LPS and IFN- ⁇ ).
  • a sufficiently sustainable signal is transmitted into the DC by a 2, preferably 4, more preferably 6-hour exposure to said maturation agents (e.g. LPS and IFN- ⁇ ) so that after said exposure the DC is irreversibly committed to complete the process of maturation and no longer depends on the presence of the ligands, i.e. DC maturation agents.
  • the DCs have not yet completed their maturation process, which takes 1-2 days.
  • the smDC1 design takes optimal advantage of the immune stimulatory time window during the first 24 hours after initiation of maturation and before the immune suppressive time window opens and starts to down-modulate the immune response.
  • DCs possess strong immune activating properties (the activating window), whereas at later stages of their development they enter an immune suppressive phase (the suppressive window).
  • the activating window Molecular mechanisms of T-cell activation are well studied and understood. The molecular nature of and the events initiating negative regulatory feedback loops are much less studied.
  • the design of the DC immune medicine according to the present invention aims at broadening the immune stimulatory window for enhancement of the immune activation and downscaling or closing the immune suppressive window, thus blocking negative regulatory feedback loops in DCs.
  • DCs either by over-expressing immune stimulatory genes in addition to exposing them first to DC maturation agents, such as LPS/IFN- ⁇ , or by knocking down immune-suppressive genes using RNA interference.
  • the expression of a multitude of immune stimulatory or immune suppressive genes may be modulated following the same basic principle.
  • the feasibility of this approach by over-expressing the immune-stimulatory CD40L molecule or by knocking down the immune-suppressive molecules IL-10 and IDO is shown in the example section. Combinations of over-expression and knock down may enhance the potency of a DC immune medicine but follow the same basic logic.
  • a T-lymphocyte suppressive DC immune medicine for the treatment of pathological over-activity of the immune system may be designed in analogy to the T-lymphocyte stimulatory DC immune medicine by genetically engineering a DC initially exposed to a maturation stimulus, such as LPS/IFN- ⁇ by genetically engineering the resulting smDCs to over-express immune suppressive molecules or knock down immune stimulatory molecules in the DC.
  • a maturation stimulus such as LPS/IFN- ⁇
  • partially matured smDCs are manipulated by introducing into said DCs nucleic acid molecules encoding the at least one immune stimulatory or immune suppressive molecule and/or nucleic acid molecules, preferably ribonucleic acid molecules (e.g. siRNA), to inhibit or prevent the expression of at least one immune suppressive or immune stimulatory molecule.
  • nucleic acid molecules encoding the at least one immune stimulatory or immune suppressive molecule and/or nucleic acid molecules, preferably ribonucleic acid molecules (e.g. siRNA), to inhibit or prevent the expression of at least one immune suppressive or immune stimulatory molecule.
  • the expression of immune stimulatory as well as immune suppressive molecules in DCs may be influenced or induced by various methods, whereby it is preferred to modulate said expressions by introducing nucleic acid molecules as outlined above.
  • nucleic acid molecule transfer can be achieved with lentiviral gene transfer vehicles as well as liposome-mediated transfection.
  • viral vectors such as retro viruses or adeno viruses, or non-viral vectors, such as gene gun or poly-cationic technologies, or when any other gene transfer is/are employed.
  • Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • RNA molecules can also be used. In mammalian cells, introduction of long dsRNA (>30 nt) often initiates a potent antiviral response, exemplified by non-specific inhibition of protein synthesis and RNA degradation. The phenomenon of RNA interference is described and discussed, e.g., in Bass, Nature 411:428-29 (2001 ); Elbahir et al., Nature 411:494-98 (2001 ); and Fire et al., Nature 391:806-11 (1998 ), where methods of making interfering RNA also are discussed.
  • the siRNAs sequences used in the present invention are preferably less than 100 base pairs, typically 30 bps or shorter, and are made by methods known in the art. Exemplary siRNAs according to the invention could have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 19 bps, 15 bps, 10 bps, 5 bps or any integer thereabout or therebetween.
  • the precursors for the manufacturing of immature DCs are obtained from skin, spleen, bone marrow, thymus, lymph nodes, umbilical cord blood or, most preferably, from peripheral blood.
  • the DCs used in the method according to the present invention can be directly isolated from a respective source or derived from progenitor cells.
  • the person skilled in the art knows respective methods.
  • DC precursors and immature DCs can be isolated by collecting anti-coagulated peripheral blood, haematopoietic stem cells, by leukocyte apheresis, or by preparation of buffy coats, rosetting, centrifugation, density gradient centrifugation (e.
  • a leukocyte population can be prepared, such as, for example, by collecting blood from a subject, defribrinating to remove the platelets and lysing the red blood cells.
  • DC precursors, monocytes or myeloid progenitor or stem cell are used to differentiate iDCs.
  • Monocytes can optionally be enriched from peripheral blood by, for example, taking advantage of their capacity to adhere to plastic surfaces, centrifugation through a density gradient, monoclonal antibody panning, counter flow centrifugation and the like. If the DCs obtainable by the method according to the present invention are used to treat individuals, the iDCs can be obtained from the individual to be treated or from a healthy individual HLA-matched to the individual to be treated.
  • DC progenitors can be cultured and differentiated in suitable culture media.
  • suitable tissue culture media include e.g. RPMI 1640 and DMEM.
  • the tissue culture media can be supplemented with human autologous or pooled donor serum but not serum of any bovine source, amino acids, vitamins, cytokines, such as GM-CSF and IL-4 or IL-13, or IFN- ⁇ , and divalent cations to promote differentiation of the cells.
  • the progenitor cells may be preferably cultured also in serum-free clinical grade media.
  • a typical cytokine combination used with dendritic cell culture medium comprises about 500 units/ml each of GM-CSF and IL-4 or IL-13, or IFN- ⁇ .
  • an effective amount of at least one DC maturation agent is contacted with the iDCs.
  • the at least one DC maturation agent is preferably selected from the group consisting of heat-inactivated or formalin-treated Bacillus Calmette-Guerin (BCG), preferably cell wall constituents of BCG, BCG-derived lipoarabidomannans or BCG components, lipopolysaccharide (LPS) derived from E.
  • an imidazoquinoline compound preferably an imidazoquinoline-4-amine compound, in particular 4-amino-2-ethoxymethyl-x-dimethyl-1H-imidazol [4,5- c]quinolin-1-ethanol or 1-(2-methylpropyl)-1H-imidazo [4,5-c] quinolin-4-amine, or derivatives thereof (see e.g.
  • a synthetic double-stranded polyribonucleotide preferably polyI:C, natural double-stranded RNA or RNA viruses or fragments of RNA, or synthetic analogues, or a synthetic or natural nucleic acid molecule comprising un-methylated CpG motifs.
  • TLR agonists see table 1 for a comparison.
  • LPS dendritic cell maturation agent
  • the CD40/CD40L signal may be applied in any combination with TLR agonists, inflammatory cytokines.
  • TLR agonists e.g., IL-4, IL-12, IL-12, IL-12, IL-12, etc.
  • inflammatory cytokines e.g., IL-12
  • the at least one (preferably at least 2, 3, 5, 10) dendritic cell maturation agent is preferably contacted with the dendritic cells in the presence of IFN- ⁇ .
  • the iDCs prior the genetic engineering step c) are contacted with effective amounts of at least one dendritic cell maturation agent for at least 2 hours, preferably for at least 6 hours, in particular for at least 12 hours, and for a maximum of up to 24 hours.
  • the maturation time depends on various parameters (e.g. DC maturation agent).
  • the contact time and the other parameters have to be chosen so that the iDCs mature only partially into smDCs using methods known in the art.
  • Cell surface markers can be detected in assays familiar to the art, such as flow cytometry and immunohistochemistry.
  • the cells can also be monitored for cytokine production (e.g. by ELISA, another immune assay, or by use of an oligonucleotide arrays or protein arrays).
  • the at least one molecule capable of mediating maturation of iDCs into IL-12 releasing smDCs is preferably selected from the group consisting of LPS in the presence of IFN- ⁇ in order to ready the DCs for the step of genetic engineering in order to manufacture a novel T-lymphocyte stimulatory or suppressive DC immune medicine with improved features.
  • the at least one molecule capable of enabling the DCs to maintain their immune stimulatory phenotype characterised e.g. by the secretion of IL-12 beyond the physiologic immune stimulatory window of approximately 24 hours, thus conferring to them superior features in comparison with smDCs, is CD40L, typically but not necessarily in the presence of IFN- ⁇ .
  • the at least one molecule that interferes with the expression of DC molecules that mediates T-lymphocyte suppressive activity is selected from the group consisting of interleukin 10 (IL-10) and indoleamine 2,3-dioxygenase (IDO).
  • the molecule that mediates T-lymphocyte suppressive activity may also be selected from the molecules listed in tables 2 and 3 of the example section, whereby molecules showing two-fold over-expression in the DNA micro array expression profiling data are preferred, but molecules showing a six-fold or higher over-expression are particularly preferred.
  • the iDCs are preferably loaded with at least one antigen before contacting them with the preferred LPS/IFN- ⁇ stimulus to manufacture smDCs followed by genetic engineering.
  • Antigen loading is necessary to instruct T-lymphocytes against what antigen they need to become active or which antigen they are supposed to tolerate.
  • Antigens for the charging of DCs may be derived from diseased tissue, such as tumour antigen or viral antigens from virally infected cells. They may be a fragment of or an entire dead or living microorganism.
  • An antigen may be a recombinant protein, or a synthetic peptide, a DNA-based viral or non-viral recombinant expression vector or natural or synthetic RNA coding for an antigen.
  • antigens may be environmental antigens that have triggered an immune dysfunction such as an allergy, an auto-antigen against which a pathologic autoimmune response has caused disease, or an antigen that determines organ or stem cell transplant rejection, such as MHC molecules. It is worth noting that, in case of a T-lymphocyte suppressive DC immune medicine for tolerance induction against an allogeneic transplant, loading might not be necessary, as the organ or stem cell donor DCs carry the same MHC molecules as the transplant.
  • a DC immune medicine will be designed in a way that it suppresses immunity against the allergen, transplantation antigen, or auto-antigen.
  • various methods may be used such as passive exposure that allows the DC to phagocytose the protein or peptide antigen, an antigenic protein complex, cells or cell membranes expressing antigens or antigenic peptides, texosomes, liposomes containing antigens or antigenic peptides, nucleic acids encoding antigens or antigenic peptides (possibly incorporated in plasmids or viral vectors), or total RNA from a tumour cell.
  • passive exposure that allows the DC to phagocytose the protein or peptide antigen, an antigenic protein complex, cells or cell membranes expressing antigens or antigenic peptides, texosomes, liposomes containing antigens or antigenic peptides, nucleic acids encoding antigens or antigenic peptides (possibly incorporated in plasmi
  • Such vehicles may be of viral or non-viral origin or may be nano-particles.
  • Antigens may be tumour antigens, viral antigens, bacterial antigens, etc., more generally, any peptide or polypeptide against which an immune response or reaction is sought.
  • DCs may be sensitised to one or several antigens according to various techniques known in the art.
  • the term "sensitized” indicates that the antigen or a portion thereof is exposed at the surface of the DCs, preferably in complex with molecules of the major histocompatibility complex (MHC). Essentially, only the presence of the antigen and its presentation to a T-lymphocyte determines the DC immune medicine but not the way the antigen reaches the DC.
  • MHC major histocompatibility complex
  • the antigen-loaded and genetically engineered DC of the present invention may be used to therapeutically modulate immune responses in various immunological dysfunctions depending on the antigen loaded into said cells.
  • Such dysfunctions may include but are not limited to cancer, a failure of the immune system to reject transformed and mutated cells; infectious disease, for example in the context of severe and otherwise untreatable microbial infections or in immune-compromised individuals, particularly during organ or stem cell transplantation.
  • Other immune dysfunctions that may be treated by such a DC immune medicine may result from immunological hyper-activity, for example against environmental antigens resulting in allergies, or in situations where the immune system attacks its host causing autoimmune diseases.
  • a DC immune medicine may be designed based on the methods of the present invention that interferes with the rejection of an organ or stem cell transplant thus facilitating the acceptance of the graft by its host.
  • the at least one antigen is selected from the group consisting of tumour antigens, viral antigens, and bacterial antigens.
  • the genetically engineered DCs according to the present invention may be loaded with any antigen against which an immune response in an individual should be induced, suppressed, or prevented. Particularly preferred are tumour antigens.
  • novel genetically engineered DC immune medicine with improved T-lymphocyte stimulatory or suppressive capacity can be preserved, e.g. by cryopreservation either before maturation as iDCs, following partial maturation as smDCs, or after genetic engineering as improved DCs prior to administration to a patient.
  • Cryopreservation agents which can be used include but are not limited to dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol,i-erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, amino acids, methanol, acetamide, glycerol monoacetate and inorganic salts.
  • DMSO dimethyl sulfoxide
  • glycerol polyvinylpyrrolidone
  • polyethylene glycol albumin
  • dextran sucrose
  • sucrose ethylene glycol,i-erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose
  • choline chloride amino acids
  • methanol
  • a further aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the novel genetically engineered DC immune medicine with improved T-lymphocyte stimulatory or suppressive capacity according to the present invention.
  • the DCs of the present invention can be formulated with physiologically acceptable carriers, excipients, buffers, and/or diluents using methods and compositions well known to the skilled artisan.
  • the novel genetically engineered DC immune medicine with improved T-lymphocyte stimulatory or suppressive capacity can be administered directly to a subject in need of immune modulation.
  • about 10 2 to about 10 10 cells are suspended in a pharmaceutically acceptable carrier.
  • the cells are preferably injected into a disease free lymph node, preferably into the inguinal region but any tumour free lymph node will serve the purpose, into the tumour directly or into a region, near to, adjacent to, or in circulatory or lymphatic contact with the tumour or tumour bed.
  • the DC immune medicine may be applied subcutaneously or intradermally into the skin to allow migration into lymph nodes.
  • Implanted release devices may be used that deliver a continuous stream of the DC medicine into the tumour or a metastasis, a lymph node, the blood stream, or the skin.
  • novel genetically engineered DC immune medicine with improved T-lymphocyte stimulatory or suppressive capacity of the present invention can be administered by any means appropriate for the formulation and mode of administration.
  • the cells can be combined with a pharmaceutically acceptable carrier and administered with a syringe, a catheter, a cannula, and the like.
  • the cells can be formulated in a slow release matrix.
  • the formulation can be administered by a means appropriate for the matrix used.
  • Other methods and modes of administration applicable to the present invention are well known to the skilled artisan.
  • compositions of the present invention can be used alone in the treatment of an individual, or the compositions can be used in combination with any other method to treat a tumour.
  • the methods of the present invention can be used in combination with surgical resection of a tumour; prior to, simultaneous with, or subsequent to radiation therapy and/or chemotherapy (cytotoxic drugs, apoptotic agents, antibodies, and the like); cryo-therapy; brachy-therapy; other forms of immune therapy (ex vivo expanded tumour antigen specific T-lymphocytes, NK cells, cytokines and growth factors, antibodies specific for tumour antigens, or targeting structures of the tumour tissue that are critical for tumour cell survival, such as blood vessels, etc.); gene therapy using viral or non-viral vectors, and the like.
  • the DC immune medicine of the present invention can be co-administered with another agent, which agent acts as an adjuvant to the maturation of the dendritic cell and/or the processing of antigen within the tumour or region near or adjacent to the tumour. Any and all of these methods can also be used in any combination. Combination treatments can be concurrent or sequential and can be administered in any order as determined by the treating physician.
  • Another aspect of the present invention relates to the use of a dendritic cell according to the present invention for the manufacture of a medicament to treat and/or prevent cancer and/or microbial or parasitic infections; or to treat and/or prevent allergies, autoimmune disease, or stem cell or organ transplant rejection.
  • the partially matured dendritic cells according to the present invention may be preferably employed in cancer prevention and/or cancer treatment. In such a case the dendritic cells are loaded with at least one tumour antigen.
  • the cells can be administered directly into a tumour, into the tumour bed subsequent to surgical removal or resection of the tumour, peri-tumorally, into a draining lymph node in direct contact with the tumour, into a blood vessel or lymph duct leading into, or feeding a tumour or organ afflicted by the tumour, e. g., the portal vein or a pulmonary vein or artery, and the like
  • the administration of the partially mature dendritic cells of the invention can be either simultaneous with or subsequent to other treatments for the tumour, such as chemotherapy or radiation therapy. Further, the partially mature dendritic cells of the invention can be co-administered with another agent, which agent acts as an adjuvant to the maturation of the dendritic cell and/or the processing of antigen within the tumour or region near or adjacent to the tumour.
  • the dendritic cells can also be formulated or compounded into a slow release matrix for implantation into a region in or around the tumour or tumour bed such that cells are slowly released into the tumour, or tumour bed, for contact with the tumour antigens.
  • the medicament is administered to an individual prior to, simultaneous with, or subsequent to radiation therapy and/or anti-tumour or anti-microbial chemotherapy, or any therapy aimed at treating allergies, autoimmune diseases, or stem cell or organ transplant rejection.
  • the dendritic cells according to the present invention may be employed in combination with other cancer therapies in order to achieve an even more beneficial effect.
  • Another aspect of the present invention relates to the use of a dendritic cell according to the present invention for the manufacture of a medicament to treat and/or prevent immunological disease caused by a pathologic over-reaction of the immune system against environmental antigens, such as allergens, or against autoantigens in the course of an autoimmune disease.
  • Said medicament is preferably administered to an individual prior to, simultaneous with, or subsequent to other modalities aimed at treating or preventing allergies or autoimmune disease.
  • a further apsect of the present invention relates to the use of a dendritic cell according to the present inivention for the manufacture of a medicament to treat and/or prevent the immunologic rejection of an allogeneic stem cell transplant, preferably used in the treatment of haematological malignancies, or to treat and/or prevent rejection of an allogeneic organ transplant.
  • Said medicament is preferably administered to an individual prior to, simultaneous with, or subsequent to other modalities aimed at treating or preventing the rejection of an allogeneic stem cell or organ transplant.
  • Fig. 1 shows developmental plasticity of a DC in a schematic representation of the kinetics of a DC differentiation process.
  • Fig. 2 shows a quality control of the smDC1 basic design.
  • Fig. 3 shows the results of a CD40L gene transfer.
  • Fig. 4 shows the quantity and quality of IL-12 and IL-10 secretion.
  • Fig. 5 shows the potential for cytolytic activity (square, CD40L transgenic DCs, GFP transgenic DCs, control DCs.
  • Fig. 6 shows the immune stimulatory capacity of LPS-activated DCs blocked for IL-10 expression.
  • Fig. 7 shows the immune stimulatory capacity of LPS-activated DCs with silenced IDO expression.
  • Fig. 8 shows DC expression profiling.
  • Leukocytes were collected using an Amicus leukocyte apheresis device (Baxter, Deerfield, IL) from healthy volunteers and patients suffering from various neoplasias treated in the context of clinical trials that were approved by the responsible institutions review boards. All individuals gave their informed consent to these studies according to the World Medical Association Declaration of Helsinki. Cell numbers and subsets were determined on a Sysmex cell counter (Sysmex, Bornbarch, Germany) and/or by flow cytometry.
  • Sysmex cell counter Sysmex, Bornbarch, Germany
  • Monocytes were enriched by plastic adherence as described previously using AIM-V (Invitrogen, Carlsbad, CA) supplemented with 1% human pooled AB plasma (Octaplas, Octapharma, Vienna, Austria) or CellGro medium (CellGenix, Freiburg, Germany). For the in-line procedures we followed the instructions provided by the manufacturers. Using the Elutra cell separator (Gambro BCT, Lakewood, CO), monocytes were enriched from the leukocyte apheresis product by loading into the elutriation chamber while maintaining the centrifuge speed at 2400 rpm.
  • Leukocyte apheresis and monocyte enrichment products were analysed for total leukocytes, T-lymphocytes, B-lymphocytes, monocytes, and granulocytes by antibody labelling with anti-CD45-FITC, anti-CD3-PerCP, anti-CD19-APC, anti-CD14-APC, and anti-CD15-FITC (BD Pharmingen San Diego, CA), respectively, using the Trucount system (Becton Dickinson, New Jersey, NJ). Labelled cells were analysed on a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA). The appropriate isotype control antibodies were included in the analysis.
  • Monocytes isolated by the respective enrichment procedures described above were cultured at a density of 1x10 6 monocytes/cm 2 either in AIM-V medium supplemented with 2% pooled human AB plasma or in CellGro medium at 37°C in a humidified incubator for 6 days.
  • the culture medium was supplemented with 1000 U/ml human GM-CSF and 300 U/ml human IL-4 (both from CellGenix, Freiburg, Germany) and replaced with the same volume of AIM-V/2%OP or CellGro plus GM-CSF and IL-4 on day 3.
  • Maturation was carried out on day 6 by adding 50 ng/ml IFN- ⁇ (Boehringer Ingelheim, Vienna, Austria) and lipopolysaccharide (LPS, E. coli strain O111:B4, Calbiochem, San Diego, CA, USA), ranging from 1-1000 ng/ml, to the culture for 6 hours to generate semi-mature (sm) DCs that subsequently were frozen; patient's DC vaccines were manufactured with clinical grade LPS (US Pharmacopeia, Bethesda, MD).
  • the maturation status of the DCs was determined using the following antibodies: anti-CD86-APC (BD Pharmingen, San Diego, CA), anti-CD80-PE (Immunotech, Beckman Coulter, Fullerton, CA), anti-CD83-APC (all three from BD Pharmingen, San Diego, CA), anti-MHC I-PE, anti-MHC II-FITC (both from Dako Cytomation, Carpinteria, CA), and anti-CD45-PerCP (BD Pharmingen, San Diego, CA).
  • the viability of the DCs was measured by propidium iodide staining (Sigma, St. Louis, MO). Cells were analysed using a FACS Calibur flow cytometer. The appropriate isotype control antibodies were included in the analysis.
  • IL-12 concentrations in the supernatant of the DC cultures were measured as described previously.
  • Allogeneic responder PBMCs were isolated by gradient centrifugation from peripheral blood. Stimulating DCs (10000, 2000, or 400) were placed in triplicates with 10 5 responder cells in 200 ⁇ l AIM-V medium supplemented with 2% pooled human plasma on a 96 well round bottom plate. For a positive reference, 10 5 responder cells were stimulated with 100 ng/ml Staphylococcal enterotoxin A/B (SEA/SEB, Toxin Technologies Inc., Sarasota, FL). On day 4 the co-culture was incubated for another 18 hours with 1 ⁇ Ci of tritium thymidine solution (NEN Life Science Products, Boston, MA).
  • SEA/SEB Staphylococcal enterotoxin A/B
  • PBMCs were harvested with a Skatron (Lier, Norway) harvester. The incorporated tritium thymidine was counted using a Trilux ⁇ -plate reader (Wallac Oy, Turku, Finland).
  • allogeneic PBMCs were labelled with CFSE (Molecular Probes, Eugene, OR) and mixed with DCs in a ratio of 1/5, 1/10, 1/20, 1/40, and 1/80. For the controls, no DCs or SEA/SEB was added.
  • PBMCs were labelled with anti-CD3-PerCP and analysed using a FACS Calibur flow cytometer. The percentage of CD3 positive CFSE negative T-lymphocytes was determined.
  • lentiviral particles were generated by co-transfection of 293FT producer cell line with pLP-plasmids encoding for viral structural proteins, polymerase and reverse transcriptase (pLP/VSVG, pLP-1, pLP-2) and plasmids containing GFP or CD40L. 72 hours after co-transfection, the whole supernatant was harvested and 100x concentrated by ultra-centrifugation. DCs were cultivated and matured under the conditions outlined above. DCs were harvested 48 hours or 6 hours after initiation of maturation, respectively.
  • Pre-matured smDCs were then transduced with lentiviral particles (250 ⁇ l 100x concentrated lentiviral supernatant/1x10 6 DC) in combination with 6 ⁇ g/ml Polybrene (from Sigma-Aldrich) plus IL-4, GM-CSF, and IFN- ⁇ in standard concentrations.
  • lentiviral particles 250 ⁇ l 100x concentrated lentiviral supernatant/1x10 6 DC
  • Polybrene from Sigma-Aldrich
  • IL-4, GM-CSF, and IFN- ⁇ in standard concentrations.
  • IL-12 quality control supernatant was taken after 24 hours, and expression of GFP/CD40L was measured after 48 hours following standard procedures.
  • DCs are manufactured according to the standard procedures outlined above. On day 6, 10 6 DCs are transfected with 100 pmol gene-specific siRNA using a lipid-based transfection reagent (Dharmacon) according to the manufacturer's instructions. Twelve hours after transfection, DCs are stimulated with LPS/IFN- ⁇ for 6 hours. All uses are in analogy to the methods outlined above.
  • DC immune medicines currently in use employ monocyte-derived DCs that are charged with an antigen of any nature, as outlined in the introduction, and exposed to a maturation stimulus that has the capacity to trigger the release of IL-12 from the DC.
  • a DC phenotype characterised by IL-12 secretion has the capacity to induce a type 1 polarisation of the immune system that supports cytolytic immunity. This implies that a stimulatory DC immune medicine needs to be applied to the patient during the time window of IL-12 secretion to allow presentation of the antigens from the DCs to the T-lymphocytes in the presence of IL-12 ( Fig. 1 ).
  • the novel genetically engineered DC immune medicine with improved T-lymphocyte stimulatory or suppressive capacity overcomes this limitation, as the genetic manipulations of the DCs allow the immune stimulatory window to remain open for a longer time period.
  • Fig. 2 shows the quality control of the smDC1 basic design that is used for genetic engineering.
  • the DC immune medicine has to meet defined quality control criteria.
  • Panel A shows the purity, viability, and yield of DCs manufactured from peripheral blood monocytes. Such monocytes are collected by leukocyte apheresis, and monocytes are enriched by counter flow centrifugation (elutriation). In the presence of IL-4 and GM-CSF, monocytes differentiate in vitro within six days into iDCs. The iDCs are charged with antigen and subsequently exposed to a maturation stimulus comprised of LPS and IFN- ⁇ for 6 hours and frozen.
  • DCs are subjected to an alloMLR potency test (panel D) by co-cultivation with CFSE-labelled allogeneic PBMCs at the indicated ratios, which triggers cell division that is associated with a dilution of the CFSE and a reduction of fluorescence.
  • panel D The bar graphs show the mean ⁇ SEM percentage of proliferating cells from three individuals.
  • the initial stimulus is also necessary for the initiation of the immune suppressive feedback loops.
  • the stronger the activation in response to a specific stimulus the stronger the feedback signalling will be in order to down-modulate the immune activation to its baseline level.
  • the maturation stimulus LPS/IFN- ⁇ results in the highest amounts of IL-12 release, but also in the highest amounts of IL-10 release.
  • Table 2 Specifications of the basic design of smDC1 for cancer vaccination.
  • DCs were genetically engineered to over-express CD40L.
  • This molecule is normally expressed from activated T-lymphocytes and interacts with CD40 on DCs transmitting a critical activating signal into the DC.
  • This experiment was designed as an example of the transfer of an activating molecule into the DC immune medicine. In principle, however, an identical procedure may be used for other stimulatory molecules or, in order to design a suppressive DC immune medicine, immune suppressive molecules may be over-expressed from a DC.
  • the rationale for CD40L gene transfer into DCs was
  • Fig. 3 shows a CD40L gene transfer.
  • panel A the expression of GFP or CD40L after lentiviral gene transfer in 6 hours smDCs and 48 hours mDCs is shown. All measurements shown here were done 48 hours after exposure of pre-matured DCs to the lentiviral vector. Expression of CD40L from DCs caused enhanced secretion of IL-12 compared to iDCs, DCs exposed to LPS/IFN- ⁇ alone, or GFP engineered 6 hours smDCs and 48 hours mDCs (panel B).
  • the enhanced IL-12 release upon GFP gene transfer is probably caused by the viral double-stranded RNA that signals via TLRs (see table 1) that are expressed in 6 hour-smDC but not any more in 48 hour-LPS/IFN- ⁇ mDCs.
  • the expression profile of functionally important DC membrane molecules (panel C) was un-altered by lentiviral gene transfer into DCs (black histograms, immature DCs; white histograms, mDCs).
  • the secretion of the cytokines IL-12 and IL-10 was qualitatively and quantitatively different in DCs that were exposed to LPS/IFN- ⁇ , CD40L/IFN- ⁇ , or a combination of both ( figure 4 ).
  • the secretion of IL-12 was almost twice as high when the combination stimulus was applied, compared to the CD40L/IFN- ⁇ stimulus alone, and also considerably higher compared to the LPS/IFN- ⁇ stimulus alone.
  • IL-12 secretion from DCs exposed to the LPS/IFN- ⁇ /CD40L combined stimulus was already clearly detectable at considerable amounts after 12 hours, whereas LPS/IFN- ⁇ and CD40L/IFN- ⁇ triggered biologically relevant levels of IL-12 secretion only between 12 and 24 hours after exposure to the initial maturation signal.
  • This observation is in line with the goal of the present invention of broadening the immune stimulatory window of DC differentiation in order to improve the stimulatory capacity of a DC immune medicine.
  • the maximum expression of IL-10 was similar when DCs were exposed to LPS/IFN- ⁇ /CD40L or CD40L/IFN- ⁇ alone, but were lower when only LPS/IFN- ⁇ was used for DC maturation.
  • the immune suppressive cytokine IL-10 was already detectable after 12 hours at biologically relevant levels after CD40L/IFN- ⁇ signalling, whereas the combination stimulus LPS/IFN- ⁇ /CD40L showed kinetics similar to those of only LPS/IFN- ⁇ matured DCs.
  • Early release of IL-10 as after CD40L/IFN- ⁇ stimulation negatively interferes with the immune stimulatory window of DC differentiation and should, in the case of designing an immune stimulatory DC medicine, be avoided.
  • a phase in which the DC can activate T-lymphocytes (characterised by IL-12 secretion) and a second phase, in which the DC will suppress the activity of T-lymphocytes (characterised by IL-10 secretion and tryptophan depletion by the activity of the enzyme IDO), will be triggered by contacting iDCs with an adequate maturation stimulus, such as LPS/IFN- ⁇ ( figure 1 ).
  • an adequate maturation stimulus such as LPS/IFN- ⁇ ( figure 1 ).
  • Fig. 4 shows the quantity and quality of IL-12 and IL-10 secretion.
  • DCs were exposed to the indicated maturation stimuli in the presence of IFN- ⁇ .
  • concentrations of IL-12 and IL-10 in the culture supernatant were measured at the indicated time points.
  • T-lymphocytes have the capacity, via type 1 polarisation of an immune response, to trigger cytolytic immunity.
  • Fig. 5 the potential of T-lymphocytes exposed to CD40L transgenic DCs to trigger cytolytic immune responses by analysing the content of granzyme B in CD8 positive CTLs was further investigated ( Fig. 5 ). Indeed, it was found that co-cultivation of CTLs with CD40L transgenic DCs resulted in clearly enhanced expression of granzyme B compared to control GFP transgenic DCs or un-transduced mDCs. This is a strong indicator of the improved cytolytic potential of such CTLs and thus provides evidence that CD40L expression from a DC immune medicine has improved immune stimulatory capacity.
  • Fig. 5 shows the potential for cytolytic activity.
  • the total percentage of CTLs was only slightly increased when PBMCs were co-cultivated with CD40L transgenic DCs (left panel, squares) compared to GFP transgenic DCs (diamonds) and un-transduced mDCs (triangles).
  • CD40L transgenic DCs left panel, squares
  • GFP transgenic DCs diamonds
  • mDCs un-transduced mDCs
  • mDCs 6 hour-LPS-matured DCs
  • CD4+, CD8+, (panel A) and CD4+CD25+FoxP3+ T-cells (panel B) were analysed on day 6 of co-cultivation using the Trucount system and a FACS LSRII flow cytometer.
  • the immune phenotype as well as IL-10 and IL-12 secretion were measured 48 hours after LPS/IFN- ⁇ activation by flow cytometry and ELISA, respectively (panel C).
  • the immune phenotypic analysis compares LPS/IFN- ⁇ -activated DCs (white histogram) with iDCs (black histogram) in IL-10-silenced DCs (panel D) or control-silenced DCs (panel E).
  • Fig. 7 shows the immune stimulatory capacity of LPS/IFN- ⁇ activated DCs with silenced IDO expression.
  • Fig. 8 shows DC expression profiling.
  • DCs were exposed to the indicated maturation stimuli or were left immature.
  • RNA was extracted at the indicated time points and subjected to expression profiling using whole genome DNA micro arrays.
  • the results of the expression profiling was analysed using CarmaWeb (Comprehensive R based Microarray Analysis, Bioinformatics Graz and the Tyrolean Cancer Research Institute, Austria). All data were grouped into 20 clusters that used the basic algorithm of the CarmaWeb software platform and identified the clusters that contained IDO and IL-10.
  • the genes in these clusters have an expression profile similar to that of the two known immune suppressive DC molecules, which led to the conclusion that they have a function in the immune regulation of a DC that is also immune suppressive (tables 3 and 4).
  • IDO expression cluster According to the CarmaWeb algorithm, the genes listed have an expression profile that resembles that of IDO (gene name INDO, bold and italics) suggesting a function similar to that of IDO (numbers are log with base 2 relative to immature DCs). 6 hours LPS IFN- ⁇ vs . 12 hours LPS CD40L IFN- ⁇ vs. 12 hours 24 hours LPS CD40L IFN- ⁇ vs. 24 hours 48 hours LPS CD40L IFN- ⁇ vs.
  • the genes listed have an expression profile that resembles that of IL-10 (gene name IL10, italics and bold) suggesting a function similar to that of IL-10 (numbers are log with base 2 relative to immature DCs). 6 hours LPS IFN- ⁇ vs. 12 hours LPS CD40L IFN- ⁇ vs. 12 hours 24 hours LPS CD40L IFN- ⁇ vs. 24 hours 48 hours LPS CD40L IFN- ⁇ vs.
  • a DC immune medicine may be modulated by genetic engineering. It was demonstrated that over-expression of immune stimulatory molecules in DCs as well as knock down of immune suppressive molecules results in enhanced immune stimulatory capacity. This may find an application as a DC cancer vaccine or an anti-infectious DC immune medicine, in which the DCs are charged with tumour derived antigens or antigens derived from microbes, exposed to a maturation stimulus, and engineered as described. Additionally, the data presented here imply that an immune suppressive DC medicine may be designed by knocking down immune stimulatory molecules and by over-expression of immune suppressive molecules. Such a suppressive DC immune medicine may have applications in allergy or auto-immunity, but also in transplantation medicine, in order to tolerise the transplant recipient's immune system to the transplanted tissue.

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EP07450233A EP2072617A1 (en) 2007-12-12 2007-12-12 Method for producing dendritic cells
AU2008334914A AU2008334914B9 (en) 2007-12-12 2008-12-12 Method for producing dendritic cells
JP2010537327A JP5730577B2 (ja) 2007-12-12 2008-12-12 樹状細胞の作製方法
CN200880120431.4A CN101896601B (zh) 2007-12-12 2008-12-12 用于生成树突细胞的方法
EP08859543.4A EP2222836B1 (en) 2007-12-12 2008-12-12 Method for producing dendritic cells
CA2709209A CA2709209C (en) 2007-12-12 2008-12-12 Method for producing dendritic cells
KR1020107011455A KR101590199B1 (ko) 2007-12-12 2008-12-12 수지상 세포를 생산하는 방법
MX2010006434A MX2010006434A (es) 2007-12-12 2008-12-12 Metodo para producir celulas dendriticas.
US12/747,759 US9303246B2 (en) 2007-12-12 2008-12-12 Methods for producing dendritic cells with an siRNA that interferes with MAPKAPK2 expression
NZ585898A NZ585898A (en) 2007-12-12 2008-12-12 Method for producing semi-mature dendritic cells and uses thereof
BRPI0819936-1A BRPI0819936A2 (pt) 2007-12-12 2008-12-12 Preparação farmacêutica, método para produzir células dendríticas, células dendríticas, composição farmacêutica, e, uso de uma célula dendrítica ou uma preparação.
PCT/EP2008/010606 WO2009074341A1 (en) 2007-12-12 2008-12-12 Method for producing dendritic cells
EA201000972A EA023106B1 (ru) 2007-12-12 2008-12-12 Способ получения частично зрелых дендритных клеток, обладающих повышенной экспрессией интерлейкина il-12
ZA2010/03193A ZA201003193B (en) 2007-12-12 2010-05-06 Method for producing dendritic cells

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EP2547360A1 (en) * 2010-03-15 2013-01-23 The Trustees of The University of Pennsylvania System and method of preparing and storing activated mature dendritic cells
EP2547360A4 (en) * 2010-03-15 2013-11-20 Univ Pennsylvania SYSTEM AND METHOD FOR THE PRODUCTION AND STORAGE OF ACTIVATED TIRES OF DENDRITIC CELLS
AU2011227447B2 (en) * 2010-03-15 2016-04-14 The Trustees Of The University Of Pennsylvania System and method of preparing and storing activated mature dendritic cells
EP3424522A1 (en) * 2010-03-15 2019-01-09 The Trustees Of The University Of Pennsylvania System and method of preparing and storing activated mature dendritic cells
KR20200062188A (ko) * 2017-09-05 2020-06-03 메디진 이뮤노테라피스 게엠바하 수지상 세포 효능 분석

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CN101896601A (zh) 2010-11-24
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EP2222836B1 (en) 2017-02-22
KR101590199B1 (ko) 2016-01-29
CA2709209C (en) 2019-05-21
JP5730577B2 (ja) 2015-06-10
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WO2009074341A1 (en) 2009-06-18
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CN101896601B (zh) 2014-09-03
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AU2008334914B2 (en) 2013-08-01
MX2010006434A (es) 2011-05-02

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